NMDA receptors (NMDARs) mediate a slow, Ca2+ permeable component of excitatory synaptic transmission, and play a central role in normal processes that are essential for life. In addition, a large number of neurological conditions either involve aberrant NMDAR function or the modulation of NMDAR function could be therapeutically beneficial. For these reasons, there is resurgent interest in the pharmacology of the NMDAR, with the discovery of multiple classes of subunit-selective modulators that act at 6 different modulatory sites on the receptor. This is in contrast to the preceding decade, during which NMDAR drug development was curtailed after multiple NMDAR antagonist trials for stroke failed, in part, due to factors unrelated to efficacy. These new modulator classes show a wide range of GluN2-subunit selectivity and diverse mechanisms, which include modulation of channel open probability, agonist potency, and deactivation time course, as well as a dependence on agonist binding. We have discovered two series of allosteric modulators that alter single channel conductance and ionic selectivity and block, establishing a new precedent in ion channel modulation. Analogous to regulation of biased signaling in GPCRs, our recent data suggest that we can regulate different facets of NMDAR function (current flow, Ca2+ permeation, Mg2+ block) through these new classes of modulators. Both classes of modulators have structural determinants located in the GluN1 pre-M1 helix, a gating motif that makes contact with the transmembrane M3 helix, which forms the channel gate. Moreover, there is a unique structure- activity relationship for the effect of modulators on ion permeation, as substitution at different positions on a phenyl ring can tune the relative proportion of subconductance levels. A deeper understanding of these properties might allow the development, for example, of NMDAR potentiators that do not trigger Ca2+- mediated neurotoxicity or biased modulators that do not alter response time course but reduce Ca2+ permeability. Such allosteric modulators should be free from on-target side effects yet may be neuroprotective for vulnerable regions, such as dopaminergic neurons in Parkinson?s patients. Given the potential utility of these novel modulators, we have designed 4 experiments that use medicinal chemistry, molecular biology, electrophysiology, and Ca2+ imaging to explore the site and mechanism of regulation of channel conductance and ionic selectivity. Aim 1: Do other classes of modulator acting on pre-M1 region alter single channel conductance? Aim 2: What is the structure-activity relationship for control of conductance and ionic selectivity? Aim 3: What are the key residues that control the different effects of these modulators? Aim 4: Do modulator-induced changes in conductance and Ca2+ permeability alter synaptic signaling?